mouse anti-syt1 antibody cat# 105 311  (Synaptic Systems)

Journal: bioRxiv

Article Title: Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1

doi: 10.1101/2024.09.12.612660

Figure Lengend Snippet: A. Schematic structure of Syt1 with its polybasic patches, marked as purple discs. The C2A and C2B domains are rendered using bioRender ( bioRender.com ) from the Protein Data Bank entries, 3F04 and 1K5W, respectively. The rest of the molecule is schematically drawn using bioRender ( bioRender.com ). B . Immunofluorescence of BON cells using anti-Syt1 antibodies. Cell nuclei were labeled with Hoechst dye (blue). Syt1 immunofluorescence (green) appears punctate, consistent with the distribution of DCVs. C . An enlarged view of the red boxed region in B, showing a cluster of puncta. D . No immunofluorescence against Syt7 could be detected in BON cells. E . As a positive control, HEK293T cells transiently expressing Syt7 displayed robust Syt7 immunofluorescence.

Article Snippet: They were then incubated overnight at 4°C with either mouse anti-Syt1 antibody (1:1000; 105.011, Synaptic Systems) or mouse anti-Syt7 antibody (1:1000; MA5-27654, Synaptic Systems,).

Techniques: Immunofluorescence, Labeling, Positive Control, Expressing

Journal: bioRxiv

Article Title: Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1

doi: 10.1101/2024.09.12.612660

Figure Lengend Snippet: A . Domain structures of the Syt1 wild-type and mutant rescue constructs used. B . Western blot (WB) analysis of the constitutive expression of syt1 transgenes in BON cells. A representative result from 3 separate experiments is shown. Scr (scrambled, mock shRNA), KD (knock-down 2), WT (pH-Syt1 WT ), K189 (pH-Syt1 K189–192A ), K326 (pH-Syt1 K326,327A ). The rescue constructs are stably expressed in BON cells constitutively expressing shRNA against syt1 (KD). The rescue constructs with pHluorin migrate slower, allowing relative amounts of expression from native vs. exogenous loci (see C). C. Quantification of the expression of endogenous syt1 vs pH-syt1 rescues, from densitometry analysis of blots as in B. (n=3 blots). 50-60 % of endogenously expressed syt1 is replaced by the expression of pH-syt1 rescue constructs, for a total amount 0.9-1.45 times the amount in the parental cell line. D. Calcium-dependent bulk release of serotonin (5-HT) from BON cells with the genotypes as indicated, from 3 independent experiments. For each genotype, the fraction of total cellular 5-HT released upon stimulation (ionomycin 10 µM, ∼3 s) was calculated, then normalized to release from the parent cell line (src). The error bars represent ± SEM in both C and D. E. Exogenously expressed pH-Syt1 is correctly targeted to DCVs. Confocal images of BON cells stably co-expressing the granule marker NPY-mCherry and pH-Syt1 constructs as indicated. After fixation, cells were permeabilized and pHluorin-Syt1 was detected using Alexa488 labeled anti-Syt1 antibodies. The boxed regions are shown at higher magnification below each panel. The Pearson correlation coefficient was coefficient of 0.718 ± 0.037 (SEM, n=4 images), indicating good co-localization (a value of 1 corresponds to perfect colocalization).

Article Snippet: They were then incubated overnight at 4°C with either mouse anti-Syt1 antibody (1:1000; 105.011, Synaptic Systems) or mouse anti-Syt7 antibody (1:1000; MA5-27654, Synaptic Systems,).

Techniques: Mutagenesis, Construct, Western Blot, Expressing, shRNA, Knockdown, Stable Transfection, Marker, Labeling

Journal: bioRxiv

Article Title: Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1

doi: 10.1101/2024.09.12.612660

Figure Lengend Snippet: A . Depiction of the experiment. A carbon fibre electrode (CFE) held at 650 mV gently touches a BON cell. Stimulation is performed by pressure-driven superfusion of an ionomycin solution from a micropipette placed nearby (“stim”). The principle of detection and the oxidation reaction are shown schematically. B. Example of an amperometric trace. The cell was stimulated for 3 s by ionomycin application (black bar). Each exocytosis event results in a brief oxidation spike. The spike marked by an * is expanded in the inset. C . Bar plot of the total number of spikes (to t=23 s) averaged over the number of tested cells, including cells that did not respond to stimulation (<5 spikes). N13 is the parent BON cell line, the other symbols are as in . Error bars represent S.E.M. The number of cells tested are indicated above every bar. D . Release kinetics from experiments as in B, plotted as the cumulative number of spikes per cell. Only cells that responded to stimulation (≥5 spikes) were included. Syt1 KD impairs release, pH-Syt1-NPYmCHerry restores release. Rescue with pH-Syt1 K189–192A or pH-Syt1 K326,327A are both defective, especially in light of remaining endogenous WT Syt1. E. Release kinetics as in D, normalized to the maximum number of spikes per cell for every group. There is no detectable delay in release. F . An example of changes induced in intracellular calcium upon stimulation using the calcium indicator Fluo-4. The parental, unlabeled BON N13 cells were used for these experiments to avoid overlap with other fluorescent molecules expressed in the rescued cell lines, but otherwise the conditions were the same as for D.

Article Snippet: They were then incubated overnight at 4°C with either mouse anti-Syt1 antibody (1:1000; 105.011, Synaptic Systems) or mouse anti-Syt7 antibody (1:1000; MA5-27654, Synaptic Systems,).

Techniques: Cell Stimulation

Journal: bioRxiv

Article Title: Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1

doi: 10.1101/2024.09.12.612660

Figure Lengend Snippet: A . An electron micrograph of Syt1 KD BON cells rescued with pH-Syt1 WT (WT). B-C . Same, for rescue with pH-Syt1 K189–192A (K189, B) or pH-Syt1 K326,327A (K326, C). D. Comparison of DCV areas among the groups ( p = 0.57 for the null hypothesis that the data in each group comes from the same distribution, using the Kruskal-Wallis test). E. Distributions of shortest DCV-PM distances d , for d ≤ 300 nm, for non-stimulated cells, for the groups shown. Labels are the same as in A-C. F . Changes in the DCV-PM distances upon a brief, ∼3s stimulation. The distributions before stimulation are the same as in E, replotted for easier comparison. Samples were prepared from at least two independent cultures.

Article Snippet: They were then incubated overnight at 4°C with either mouse anti-Syt1 antibody (1:1000; 105.011, Synaptic Systems) or mouse anti-Syt7 antibody (1:1000; MA5-27654, Synaptic Systems,).

Techniques: Comparison

Journal: bioRxiv

Article Title: Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1

doi: 10.1101/2024.09.12.612660

Figure Lengend Snippet: A . Schematic of a single amperometric oxidation event (see ). B . Cumulative distribution function (CDF) of maximum spike amplitudes averaged over BON cells expressing Syt1 WT (WT), Syt1 K189–192A (K189), or Syt1 K326,327A (K326). Syt1 WT spikes have lower amplitude on average. See for a summary. C . Cumulative distribution of spike widths at half amplitude for BON cells expressing Syt1 WT , Syt1 K189–192A , or Syt1 K326,327A (symbols as in B). Syt1 WT spikes last longer. D. Cumulative distribution of oxidation charges for individual events for BON cells expressing Syt1 WT , Syt1 K189–192A , or Syt1 K326,327A . No significant difference is found, implying the same amount of 5-HT is released per event for the different conditions. The p -values in B-D are returned from the Kruskal-Wallis test for the null hypothesis that all data come from the same distribution. E. Example of a trace of fusion pore permeability scaled by DCV volume ( g / V ) as a function of time (blue trace). The corresponding amperometric current I ( t ) is shown in red. The integral is carried only to 60% of the total spike duration (see text and Methods). F . Mean g / V for 0-60% of spike duration, averaged over cells in each group. Error bars represent SEM. For WT, K189, and K326 groups, the number of cells (spikes) were 17 (197), 12 (108), 11 (131), respectively. Mean values for every cell were averaged across cells. p = 0.003 and 0.020 for the mean ranks of K189 and K326 data compared against WT (see methods).

Article Snippet: They were then incubated overnight at 4°C with either mouse anti-Syt1 antibody (1:1000; 105.011, Synaptic Systems) or mouse anti-Syt7 antibody (1:1000; MA5-27654, Synaptic Systems,).

Techniques: Expressing, Permeability

Journal: bioRxiv

Article Title: Vesicle docking and fusion pore modulation by the neuronal calcium sensor Synaptotagmin-1

doi: 10.1101/2024.09.12.612660

Figure Lengend Snippet: Undocked (U) DCVs reversibly tether (T) to the PM. Initial tethering is mediated by large tethering molecules such as CAPS and/or Munc13, which also assist subsequent stages of DCV maturation at the PM. Docked DCVs (D) are closer to the PM. Docking involves Syt1 and likely SNARE proteins and acidic lipids in the inner leaflet of the PM. An elevation of [Ca 2+ ] i leads to fusion (F). Release kinetics and extent depend on the amount of docked DCVs. If the docked population is sparse, the release rate may be limited by the rate of docking.

Article Snippet: They were then incubated overnight at 4°C with either mouse anti-Syt1 antibody (1:1000; 105.011, Synaptic Systems) or mouse anti-Syt7 antibody (1:1000; MA5-27654, Synaptic Systems,).

Techniques: